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. 2018 Sep 8;46(20):10724–10739. doi: 10.1093/nar/gky810

Figure 1.

Figure 1.

CDK1, Aurora A and PLK1 kinase activity is required for MMEJ. (A) DR-EGFP-HR, (B) EGFP-MMEJ reporter and modified DNA DSB induction method by RNA-guided endonuclease Cas9. (C) Drug exposure and DSB repair assay. U2OS cells carrying the indicated DSB repair reporter substrate were treated with nocodazole (330 nM) or mock treated with DMSO for 16 h followed by Dox in the presence of nocodazole to induce a DSB. After 24 h Dox induction, the cells were trypsinized and FACS was performed. (D and E) After 16 h nocodazole treatment, the cell-cycle profile was determined by FACS and the mean percentage of cells in the indicated cell-cycle phases was determined. (F) HR repair efficiency in unperturbed and synchronized cells with or without the indicated drugs was determined by FACS. A CDK1 inhibitor (10 μM RO3306), PLK1 inhibitor (10 μM BI2536), Aurora A inhibitor (2 μM MLN8054) or DMSO was added to the culture before Dox induction. (G) MMEJ repair efficiency determined by the same method and strategy as the HR assay. (H) EGFP-MMEJ assay was performed in U2OS cells with overexpressed Flag-PLK1 WT or Flag-PLK1 kinase dead (K82M/D176N) mutant. Western blotting shows the expression of Flag-PLK1 variants. (I and J) U2OS cells overexpressing Flag-PLK1-WT or a PLK1-AS (analog-sensitive) mutant were treated with or without the indicated amounts of 3MB-PP1 and then analyzed by EGFP-MMEJ assay. Western blotting shows the expression of Flag-PLK1 variants. The data represent the means of three independent experiments, with error bars as SD and P values as noted: **P ≤ 0.01; n.s. not significant.